Nematodes as model organisms for the investigation of neurodegenerative diseases, in particular parkinsons disease, uses and methods for the discovery of substances and genes which can used in the treatment of the above disease states and identification of anematode gene

ABSTRACT

The invention relates to nematodes as model organisms for the investigation of neurodegenerative diseases in particular. Parkinsons disease, uses and methods for the discovery of substances and genes which can be used in the treatment of the above disease states and identification of a nematode gene, from  C. elegans , which is homologous to the human parkin gene associated with Parkinsons disease. The invention further relates to those nematodes which contain an aberrant or missing expression of at least one gene, preferably a parkin gene and/or a α-synuclein gene, which is connected with Parkinsons disease. According to the invention, the above organisms can be used for the identification and characterisation of medicaments for the treatment of said disease states.

The invention relates to nematodes as model organisms for investigatingneurodegenerative diseases, and, in particular, Parkinson's disease,which nematodes can be used for developing pharmaceuticals for treatingneurodegenerative diseases, including those diseases in whichplaque-like deposits (amyloidoses) occur, and, in particular, fortreating Parkinson's disease.

The animal model according to the invention is based on a gene which isconnected to the development of Parkinson's disease either beingexpressed in an aberrant manner, or not being expressed at all, in anematode.

An animal model of this nature can also be used for clarifying themetabolic pathways and for identifying new genes which are involved inParkinson's disease.

BACKGROUND OF THE INVENTION

Transgenic animal models are already available, as valuable tools forclarifying disease processes and identifying and characterizingpharmaceuticals which have a prophylactic or therapeutic effect, for avariety of disease types. The prerequisite for developing such animalmodels is, in particular, knowledge of genes which are involved in thegiven disease processes.

Aside from Alzheimer's disease, Parkinson's disease is the most wellknown disease in the neurodegenerative disease group. It ischaracterized by (1) a slowing down of all movements (bradykinesia),quiet and monotonous speech (akinesia or hypokinesia), absence of thephysiological associated movements, a stooped posture, a small-step,partially shuffling gait, handwriting which becomes smaller as thewriting continues, uncontrollable disturbances in movement, with atendency to fall forward to the side or backward, (2) rigidity of themusculature (rigor), and (3) coarse resting tremor (trembling).Parkinson's disease is a disease which occurs relatively frequently anddevelops in approx. 1% of individuals aged over 60, in particular inmen. The disease is caused by loss of dopamine in the striatum,resulting in the degeneration of neurons in the substantia nigra. Theprimary reason for loss of dopamine is not known (Dunnett and Björklund,1999; Olanow and Tatton, 1999). Although the appearance of Parkinson'sdisease is sporadic as far as most patients are concerned, there is asmall group of patients in whose families the disease occurs frequently.Molecular-genetic analyses performed in these families have led to theidentification of several genes which, having been altered by mutations,are causatively involved in the onset of Parkinson's disease. One ofthese genes, which was discovered in 1998 by Nobuyoshi Shimizu,Yoshikuni Mizuno and their coworkers, has since then been designatedParkin or PARK2 (Kitada et al., 1998; Hattori et al., 1998). This genewas found to be mutated in several families in which several familymembers had developed autosomal recessive juvenile (beginning before the40^(th) year of life) Parkinson's disease. The protein of the Parkingene is characterized, inter alia, by an ubiquitin domain in the Nterminus, two RING finger-like motifs in the C terminus and an IBR (inbetween ring fingers) domain.

Another gene which has also been identified as being causativelyinvolved in Parkinson's disease is the a-synuclein gene (Polymeropouloset al., 1997). α-Synuclein is found, in particular, in the fibrillary,intracytoplasmic inclusions (Lewy bodies) which appear in associationwith Parkinson's disease. Aside from the presynaptic proteina-synuclein, a large number of proteins, such as ubiquitin andneurofilament are also represented in the Lewy bodies. Mutations (A53Tand A30P) in the α-synuclein gene on human chromosome 4q21-q22 lead toan autosomally dominant form of Parkinson's disease (Polymeropoulos etal., 1997; Kruger et al., 1998). How these mutations are able to induceParkinson's disease has still not been elucidated. The dominantinheritance pattern and the fact that α-synuclein is present in Lewybodies in fibrillary aggregations whose formation can be accelerated bythe two mutations points to toxicity as the mechanism (toxic gain offunction). A recent study has identified a sequence of 12 amino acids inthe middle of α-synuclein which is responsible for the aggregation invitro and which is absent from the very similar protein β-synuclein,which is nonaggregating (Giasson et al., 2000). α-Synuclein has thus faronly been identified in vertebrates, with threonine, instead of alanine,as is the case in humans, being found in position 53 of the amino acidsequence in all the species known to date (Clayton and George, 1998).

Transgenic animal models which express different variants of humanα-synuclein have thus far been established in the mouse and inDrosophila. The neuronal expression of α-synuclein in the mouse leads toa progressive accumulation of α-synuclein in intraneuronal inclusions,which inclusions are not, however, of a fibrillary nature (Masliah etal., 2000). By contrast, transgenic flies, which express α-synucleinpanneuronally, exhibit fibrillary inclusions, which resemble the Lewybodies, a loss of dopaminergic neurons and impairment of locomotoryfunctions (Feany and Bender, 2000). In this connection, there were nosignificant differences in the expression of the individual forms ofα-synuclein (wt, A53T and A30P).

SUMMARY OF THE INVENTION

As compared with the animal models for Parkinson's disease which havealready been published, the advantages of a nematode model, inparticular of the C. elegans model, are, in particular, its suitabilityfor a high-throughput method (high-throughput screening) (HTS), thepossibility of being able to carry out a genetic analysis more rapidlyas a result of a shorter generation time (2-3 days, as compared withweeks in the case of Drosophila) and detailed knowledge of the molecularand functional properties of the nervous system in C. elegans. Since C.elegans can be maintained in microtiter plates, it is possible to usethis test system to test out 10,000 or more substances by HTS, on theliving worm, in a short period of time.

The progressive sequencing of different genomes has shown that manyhuman genes have homologs in other organisms. More than two thirds ofall the human disease-associated genes known thus far have beendemonstrated to be also present in the C. elegans genome. In severalcases, it has been shown experimentally that these genes arefunctionally interchangeable. These features possessed in commondemonstrate that the functions of these genes are essential forfundamental biological processes. The common features make it possibleto establish these model systems for investigating human gene functions,for identifying new target genes and for developing drugs.

The present application describes, inter alia, a Parkin-encoding gene(FIG. 3; SEQ ID NO:1), which is derived from C. elegans and which waspreviously unknown, and its gene product (SEQ ID NO:2), which gene wasfound by analyzing C. elegans DNA sequences, and comparing them with theknown sequence of the human Parkin gene, using bioinformatic methods.

Having knowledge of this novel gene, and concomitantly using other genesknown to be connected with Parkinson's disease, such as the α-synucleingene, it was possible to develop a nematode model which can be used toanalyze neurodegenerative diseases genetically, an approach which can beused for selectively developing novel pharmaceuticals for preventingand/or treating diseases of this nature and, in particular, forpreventing and/or treating Parkinson's disease and for identifying otherpotential diseases of this nature and, in particular, genes which elicitParkinson's disease.

Following identification of the Parkin gene in C. elegans, strains ofthis organism in which the C. elegans Parkin gene was wholly orpartially deleted (what are termed Parkin knock-out mutants) wereisolated in the applicant's laboratory (see experimental section). Thesestrains exhibited a phenotype which was different from that of the wildtype.

This phenotype was subjected to a variety of complementationexperiments. In these experiments, a part of the cosmid which containsthe C. elegans Parkin gene and its promoter, on the one hand, and, onthe other hand, the human Parkin gene, were inserted into the C. elegansParkin knock-out strain in order to offset the phenotype of theinactivated C. elegans Parkin gene. These experiments verified, on theone hand, that the phenotype was indeed the consequence of inactivatingthe C. elegans Parkin gene and, on the other hand, that the C. elegansgene and the human disease-associated gene are functionallyinterchangeable.

Since mutations in, and deletions of, quite different regions of theParkin gene lead to hereditary Parkinson's disease in humans, it can beassumed that expression of the disease must result from a functionalloss of the Parkin gene product. We have therefore developed the C.elegans Parkin knock-out strains as model systems for testingpharmacologically active substances. In this connection,pharamacologically active substances are those which suppress thephenotype, i.e. it is consequently possible to identify substances whichsuppress the phenotype (by, for example, these substances improving themotility of the worm).

It is not always possible to use the disease-relevant gene productitself as the therapeutic target structure for the drug development. Thepossibility then presents itself of investigating the pertinentmetabolic pathway, and the gene products involved, for their suitabilityas target structures in an in vivo model. The previously mentioned C.elegans Parkin knock-out worm strains are also outstandingly suitablefor identifying new genes which are involved in the Parkin metabolicpathway. In these experiments, the knock-out worm strains are subjectedto a mutagenesis and animals which no longer exhibit an alteredphenotype, and which consequently move normally once again, are selectedout. These animals are then subjected to genetic mapping in order toidentify the gene which, having been mutated, leads to the Parkinknock-out phenotype being suppressed. This target gene may then possiblyin turn be suitable for developing a drug. Aside from the model based onthe Parkin gene, a transgenic nematode model in which a humanα-synuclein gene is expressed in C. elegans under the control ofspecific C. elegans promoters was also developed. This model alsoreproduces important features of Parkinson's disease and canconsequently also be used as a tool for developing novel medicines fortreating this disease.

Expression of the human α-synuclein gene, or of derivatives of the humanα-synuclein gene, in particular of mutants which induce hereditary formsof Parkinson's disease in humans, such as the α-synuclein mutant A53T,in which the amino acid alanine at position 53 is replaced withthreonine, in C. elegans animals led, for example, to a morphologicalchange in the head region (anterior third of the body) and/or tailregion and/or in/on the vulva (posterior third of the body) of theanimals, which change is characterized by a swelling (increase in thediameter of the animals) due to a functional incapacity or functionalreduction in the activity of muscles and neurons. Expression ofα-synuclein or its derivatives in C. elegans presumably modifies thefunction of genes, in particular those which are expressed in musclesand neurons.

Under a first aspect, therefore, the invention relates to a nemtatodewhich exhibits an aberrant or nonexistent expression of a gene which isconnected with Parkinson's disease, in particular a Parkin gene and/oran α-synuclein gene. This nematode can be used as a model organism forneurodegenerative diseases, including those in which plaque-likedeposits appear in the nervous system (amyloidoses), and, in particular,for Parkinson's disease.

In this connection, the aberrant or nonexistent expression can relate,for example, to the Parkin gene derived from C. elegans, to the humanParkin gene or to any homologous Parkin gene from another organism, orto an α-synuclein gene from any organism, such as a human α-synucleingene.

In particular, the aberrant or nonexistent expression of the Parkin genecan be caused by this gene having been completely or partially deletedor by this gene having been temporarily inactivated, for example usingthe RNA interference (RNAi) technique which is known in the prior art.

Under another particular aspect, the nematode of the invention exhibitsa phenotype which is associated with the aberrant or nonexistentexpression of the Parkin gene and/or the α-synuclein gene.

The phenotype which is associated with the aberrant or nonexistentexpression of the Parkin gene can, for example, be a defect inchemotaxis, a defect in egglaying, an extended period of development, adecreased number of descendants, problems in coordination, a defect indefacation, retarded locomotion or a reduced body length.

The phenotype which is associated with aberrant or nonexistentexpression of the α-synuclein gene can, for example, be a defect inegglaying, a defect in the formation of the vulva, deposits ofα-synuclein, an extended period of development or a decreased number ofdescendants.

The different phenotypes are described in further detail in theexperimental section.

In addition, the invention encompasses the use of a nematode of theinvention as a model organism, in particular

-   -   for investigating the mechanisms involved in the development,        cause and/or propagation of neurodegenerative diseases,        including those in which plaque-like deposits (amyloidoses)        appear in the nervous system, and, in particular, Parkinson's        disease,    -   for identifying and/or characterizing pharmaceuticals and genes        for preventing or treating neurodegenerative diseases, including        those in which plaque-like deposits (amyloidoses) appear in the        nervous system, and, in particular, Parkinson's disease, and    -   for identifying and/or characterizing active compounds and genes        which are able to modify the effect of pharmaceuticals which are        used for preventing or treating neurodegenerative diseases,        including those in which plaque-like deposits (amyloidoses)        appear in the nervous system, and, in particular, Parkinson's        disease.

Under other aspects, the invention encompasses methods for investigatingthe efficacy of a substance or a gene in the treatment and/or preventionof such diseases and methods for investigating the suitability of asubstance or a gene for modifying the effect of a pharmaceutical in thetreatment and/or prevention of such diseases, which methods use thenematodes of the invention.

In addition, the invention also extends to the substances and geneswhich are identified by the methods according to the invention and whichcan be used for producing pharmaceuticals for preventing and/or treatingsuch diseases and as lead substances and lead genes, respectively, fordeveloping substances and genes, respectively, which are derivedtherefrom and which are active in the treatment and/or prevention ofsuch diseases, or of substances and genes, respectively, which aresuitable for modifying the effect of a pharmaceutical in the treatmentand/or prevention of such diseases.

In this connection, the development of novel pharmaceuticals fortreating Parkinson's disease or other neurodegenerative diseases,including those which are characterized by the deposition of proteinaggregates in the nervous system and are, in particular, induced orintensified by such a deposition, can also encompass the investigationof protein-mediated interactions or the investigation of factors whichare affected by these interactions.

In this connection, a pharmaceutical for treating such diseases should,in particular, be suitable for curing the disease, for alleviatingindividual symptoms, several symptoms or all the symptoms of thedisease, or for delaying the progress of the disease.

A prophylactic treatment can be aimed at complete protection against thedisease developing or simply aimed at delaying the onset of the disease.

Finally, the invention relates to an isolated nucleic acid moleculewhich encodes the C. elegans Parkin gene, to homologs and fragments ofthis molecule, and to a polypeptide or protein which is encoded by sucha nucleic acid molecule.

DETAILED DESCRIPTION OF THE INVENTION

More specifically, the invention is based on the finding that genes ofthe Parkin gene family and genes of the α-synuclein gene family arebiochemically or genetically connected with the development, propagationor intensification of neurodegenerative diseases, including those inwhich plaque-like deposits appear in the nervous system (amyloidoses),i.e. those which are induced or intensified by the deposition of proteinaggregates in the nervous system, and, in particular, Parkinson'sdisease.

Accordingly, the invention is directed, in particular, to nematodes foruse as model organisms for neurodegenerative diseases, including thosein which plaque-like deposits appear in the nervous system, whichnematodes exhibit an aberrant or nonexistent expression of a Parkin geneand/or an α-synuclein gene.

However, the genes which come into consideration in this regard alsocomprise homologous genes from other organisms. Thus, a sequencecomparison based on the C. elegans Parkin gene showed that homologousgenes were present, for example, in the following organisms:

D. melanogaster: Parkin (27% identical, 38% similar) H. sapiens: PARK2(Parkin) (27% identical, 36% similar) M. musculus: Parkin (25%identical, 36% similar)

The above genes can consequently also be introduced into the modelorganism.

In connection with the invention, an aberrant gene expression isunderstood as being, in particular, at least one of the following:

-   -   an increased gene expression which leads to the generation of a        quantity of transcription product and/or translation product of        the gene concerned which is increased as compared with the        wild-type gene,    -   a decreased gene expression which leads to the generation of a        quantity of transcription product and/or translation product of        the gene concerned which is decreased as compared with the        wild-type gene,    -   an expression of a mutated gene, and/or    -   an expression of a gene which is homologous or heterologous with        respect to the organism.

In this connection, the terms “aberrant or nonexistent expression” arealways to be understood in relation to an organism which is otherwisegenetically equivalent, i.e. identical, but which exhibits a wild-typecharacter with regard to this expression. The reference organism whichis enlisted for the comparison can consequently be, for example, a purewild-type organism which is found in nature, an organism from a linewhich has been obtained by conventional breeding methods, such as bycrossing, or a transgenic organism which has already been modified bygenetic mutation, i.e. with regard to genes of another type, and which,where appropriate, can additionally have been subjected to crossing. Theonly thing which is crucial is that the reference organism or parentorganism exhibits a wild-type character with regard to the geneexpression which is relevant in the present case.

The gene which is to be expressed aberrantly within the context of theinvention can be a gene which is homologous or heterologous with respectto the target organism or be derived by mutation from a gene which ishomologous or heterologous with respect to the target organism.

The gene which is to be expressed aberrantly can be present in thetarget organism

-   -   in the complete state, i.e. together with all the appurtenant        regulatory regions in addition to the coding region(s),    -   together with only a part of the regulatory regions in addition        to the coding region(s), or    -   solely in the form of the coding regions, i.e. in combination        with regulatory regions which are heterologous with respect to        the gene.

In this connection, the coding regions can constitute the entirety ofthe coding regions of the relevant gene, or parts of these codingregions, which parts, however, at least guarantee the functionality, ofthe gene or the gene product, which is essential in connection with saiddiseases. The coding regions can also be present in the form of a fusedgene, i.e. coding regions which are fused to a different protein, forexample a marker protein.

Thus, within the context of the invention, the aberrant gene expressioncan constitute or encompass the expression of a gene which is native butheterologous with respect to the target organism.

As a rule, it will be possible to attribute increased or decreased geneexpression to (a) mutation(s) in the promoter region and/or otherregulatory regions, e.g. enhancer sequences, matrix/scaffold attachmentregions, etc., of the relevant gene which is homologous or heterologouswith respect to the target organism or to a coupling of the coding generegions to a stronger or weaker promoter which is heterologous withrespect to the coding gene region. An increased gene expression can alsobe brought about by the additional, and suitable, incorporation ofexpression-augmenting elements, such as enhancer sequences andmatrix/scaffold attachment regions, into the genes which are to beexpresssed aberrantly.

When a mutated gene product is expressed, one or more mutation(s) is/arepresent, where appropriate in addition to mutations in regulatoryregions, in the coding region of the gene which is homologous orheterologous with respect to the target organism, which mutation(s)lead(s) to a change in the amino acid sequence of the translated geneproduct as compared with the amino acid sequence of the native geneproduct.

In this connection, the simultaneous presence of mutations in regionsregulating gene expression and in coding regions should naturally alsobe included. For example, it is possible to overexpress a mutatedprotein in a target organism in this way.

In this connection, and depending on the individual case, the aberrantgene expression can, where appropriate, include the deletion of one ormore native genes in the target organism. For example, when expressing anative or mutated heterologous gene in the target organism, it could behelpful, in individual cases, to delete the homologous gene, having thesame function, in the target organism. It could be appropriate to do thesame in individual cases when expressing a mutated homologous gene in atarget organism.

Furthermore, preference may be given, where appropriate, to regulatingthe aberrant gene expression in a tissue-specific manner. For this, thegenes which are to be expressed aberrantly are as a rule attached topromoter sequences which permit tissue-specific or cell-specificexpression, for example in the nervous system, in particular in neuronsor special neuron cell types. However, enhancer sequences which act in atissue-specific manner are also known, which sequences are able, whensuitably incorporated into a gene to be expressed, to bring about atissue-specific increase in th e expression. Tissue-specific orcell-specific promoters and enhancers of this nature are known tospecialists (see, for example, Baumeister et al., 1996; Way & Chalfie,1989; and Hobert et al., 1997).

In addition or alternatively, it is possible, by means of coupling therelevant gene to an inducible promoter, to obtain an expression, of therelevant gene in a target organism, which can be induced by chemicalstimuli or physical stimuli (e.g. temperature). In this case, it is alsopossible to conceive of using development-specific promoters. In bothcases, it is consequently possible to specify the beginning of theexpression of the relevant gene, and consequently, in particular, theappearance of phenotypes which are connected to this gene expression, ata time which can be freely selected by the user (inducible promoter) orwhich can be regulated by the choice of the (development-specific)promoter. In these cases, it is accordingly possible to initially allowthe development of the target organism to proceed for a certain periodof time unaffected by the expression of the relevant gene.

The aberrant gene expression will frequently encompass the introductionof the gene, which is to be expressed aberrantly and which is in theform of a transgene which, as explained, may be homologous orheterologous with respect to the target organism, into cells of thetarget organism using a construct which comprises the transgene. In thecase of a transgene which is homologous with respect to the targetorganism, the transgene is located, after having been introduced, in agenetic environment which is different from that of the correspondinggene which is naturally present in the target organism. In thisconnection, the transgene can, after having been introduced into thecells, be present extrachromosomally in these cells and be expressedfrom this extrachromosomal construct. Alternatively, after having beenintroduced into the cell, the transgene is integrated into the genome ofthe cell and expressed as such.

A large number of standard techniques, which are, in particular,especially suited for the organism cells employed, are available to theskilled person for transforming or transfecting organism cells withconstructs which contain the relevant transgenes and, more generally,for preparing the organisms according to the invention. Protocols forthese standard techniques can be readily found in the literature andthere will therefore be no further comments in this connection.

For example, a method for generating a nematode according to theinvention which is employed as a model organism can comprise introducingan expression construct, which contains a desired transgene or a desirednucleic acid sequence, into an hermaphrodite nematode. This can beeffected, for example, by means of microinjection. After descendantshave hatched from the eggs of the nematode, they are allowed to develop,after which at least one descendant, which contains the desiredtransgene or the desired nucleic acid under the control of regulatorysequences which permit an expression which is, where appropriate,tissue-specific or cell-specific, is identified. For the purpose of thisidentification, the expressed construct which is used for thetransfection can additionally contain a marker gene which encodes areadily detectable marker protein, where appropriate as a fusionprotein.

The nematode descendant which has been identified in this way can then,if desired or necessary, be subjected, where appropriate, to a furtherbreeding procedure, for example crossing with other nematodes which, forexample, are expressing other interesting transgenes, in order toproduce lines which exhibit various desired properties in addition tothe aberrant or nonexistent gene expression discussed above.

If, in an organism according to the invention, it is desired to renderthe expression of a native gene nonexistent, several possibilities, suchas a selective knock-out mutation, which is described in more detail inthe experimental section, or the use of a specific antisense or sensestrategy, and a selective mutagenesis, are also available for thispurpose.

Another possibility of inactivating a gene is that of using the RNAinterference technique, which was established in C. elegans about twoyears ago. In this technique, double-stranded RNA derived from the geneto be analyzed is introduced into the worm. This RNA is evidently cutinto relatively small fragments and can subsequently become distributedthroughout the animal. The RNA fragments interact, in each cell, withthe corresponding messenger RNA, resulting in the transcript beingbroken down specifically (Zamore et al., 2000). This process leads to aloss-of-function mutation having a phenotype which, over the period of ageneration, comes to very closely resemble that arising from a deletionof this gene.

There are two different possibilities for transferring thedouble-stranded RNA into the worm. The first-possibility is that ofmicroinjection, with the single-stranded RNA being prepared by in vitrotranscription. Sense and antisense RNA are subsequently hybridized toeach other, giving rise to the double-stranded form. This is injectedonce into the worm and the F1 descendants of the animal are analyzed.

The second possibility is that of feeding, with the RNA beingsynthesized in vivo in a suitable bacterial strain. As a result of theworms growing on these feed bacteria, the RNA passes from the intestinaltract into the remaining cells, leading to the breakdown of thecorresponding mRNA which has already been described above. The advantageof this approach is that the RNA is supplied continuously and theinterfering effect therefore lasts longer.

In a special embodiment of the invention, more than one of theabovementioned genes is expressed aberrantly in an organism according tothe invention. In addition to this, the organism can also express aParkin gene, or a gene which is homologous to it, as a transgene and/orexpress an α-synuclein gene, or a gene which is homologous to it, as atransgene.

The organisms according to the invention are nematodes, in particularnematodes of the genus Caenorhabditis, for example C. elegans, C.vulgaris or C. briggsae.

All of the transgenic animals which are defined within the context ofthis application can be used as model organisms for neurodegenerativediseases in which, in particular, the members of the α-synuclein genefamily and/or the genes of the Parkin gene family are involved, and, inparticular, for selectively developing novel drugs for preventing and/ortreating diseases of this nature.

In this connection, it is possible to use pharmaceutical candidatesubstances, in particular small molecules, to test whether the phenotypeobserved in the given particular case is reduced or augmented by theaction of such substances. For this reason, the transgenic animals, inparticular C. elegans, and, for example, those which are expressingα-synuclein, are suitable for testing novel pharmacological activecompounds.

The use of nematodes has numerous advantages, particularly foridentifying and characterizing pharmaceuticals and active compounds.Nematodes according to the invention exhibit, in particular, symptomswhich can be observed in a quite equivalent manner in human patients whoare suffering from neurodegenerative diseases, including those in whichplaque-like deposits appear in the nervous system, and, in particular,those patients who are suffering from Parkinson's disease. In thisconnection, it is possible to readily observe or determine the symptomsin nematodes, without any great input and only a short time after thenematodes have hatched, for example only a few days after hatching. Inaddition to this, nematodes have short generation times, as compared toother organisms, and there is the possibility of geneticallymanipulating large numbers of animals and correspondingly generatinglarge numbers of transgenic animals. There are no problems in producingrelatively large numbers of identical transgenic descendants from onenematode according to the invention, which means that sufficientorganisms can be provided even for the HTS of large numbers ofpharmaceutical candidates or of whole substance libraries. Substancelibraries of this nature can be libraries of synthetically producedsubstances or libraries of natural products.

When nematodes are used, mass screening of this nature can, for example,be conveniently performed in microtiter plates, with it being possibleto provide one well, or, in the case of parallel samples,correspondingly more wells, available for each pharmaceutical candidatecompound or active substance candidate compound.

In a method for investigating the efficacy of a substance in treatingand/or preventing neurodegenerative diseases, it is accordinglypossible, for example, for organisms according to the invention to beexposed to the substance and for changes which may arise in thephenotype, which is associated with the aberrant or nonexistentexpression of the at least one gene connected to diseases of thisnature, as has been discussed above, to be determined.

In this connection, an attenuation of the observed phenotype, anabolition of the observed phenotype or a retardation in thedeterioration of the observed phenotype with time which is otherwiseobserved in the absence of the pharmaceutical treatment, indicates thatthe investigated substance is effective as a pharmaceutical in thetreatment and/or prevention of such diseases. A deterioration in theobserved phenotype over and above the extent observed in the absence ofpharmaceutical treatment, or an acceleration of the deterioration whichis normally observed in the absence of pharmaceutical treatment,indicates that the investigated substance is contraindicated inconnection with these diseases.

In order to confirm the results which have been obtained, it will alwaysbe appropriate to observe control organisms which are not exposed to thesubstance to be investigated. Since, as a rule, a deterioration in thephenotype, i.e. in the symptoms which are observed in connection withthe aberrant or nonexistent expression of the at least one geneconnected with diseases of this nature, will be observed over time inthe target organisms, it will as a rule be necessary, in comparativeinvestigations (comparative investigations using differentpharmaceutical candidate substances or comparative investigations withand without a pharmaceutical candidate substance), to use organismswhich are equivalent to each other and which are of the same age, forexample nematodes of the same line which have hatched at the same pointin time.

In a method for investigating the suitability of a substance formodifying the effect of a pharmaceutical in the treatment and/orprevention of neurodegenerative diseases, including those in whichplaque-like deposits appear in the nervous system, it may, for example,be necessary to perform the following steps:

-   -   to expose an organism according to the invention to the        pharmaceutical and to determine the changes, which arise as a        result of the effect of the pharmaceutical, in the phenotype        which is associated with the aberrant or nonexistent expression        of the at least one gene connected to such diseases, as have        been discussed above,    -   to expose at least one further specimen of the organism of the        type used in the preceding step to the pharmaceutical in the        presence of the substance and to compare any changes which may        arise in the phenotype with the changes, which arise due to the        effect of the pharmaceutical on its own, in the phenotype which        is associated with the aberrant or nonexistent expression of the        at least one gene connected to such diseases, as have been        discussed above.

The substance to be investigated can be added before, at the same timeas, or after the addition of the pharmaceutical, which is in each caseemployed, to the assay system containing the organism according to theinvention. It is also possible for the substance and the pharmaceuticalto be previously incubated together before being added to the assaysystem.

In this connection, an attenuation of the observed phenotype which isaugmented in the presence of the substance, an abolition of the observedphenotype which newly occurs in the presence of the substance, or achronological retardation, which is stronger in the presence of thesubstance, of the changes in the observed phenotype which are otherwiseobserved in the presence of the pharmaceutical without any addition ofthe substance, indicates that the investigated substance is active as aneffect-modifying agent which augments the effect of the pharmaceutical,used in the assay, in the treatment and/or prevention of such diseases(=agonistic effect). A deterioration, which is observed in the presenceof the substance, of the observed phenotype as compared with thephenotype which is observed in the presence of the pharmaceutical on itsown, a complete abolition of the effect of the pharmaceutical, or a moreminor retardation of the deterioration which is normally observeddespite the presence of the pharmaceutical, show that the investigatedsubstance is contraindicated (=antagonistic effect) in such diseases.

That which has been said in the preceding paragraphs also applies, in acorresponding manner, with regard to the reference organisms which areto be employed for comparative purposes, including organisms which arenot treated with pharmaceutical and substance and organisms which areonly treated with the substance, i.e. which are not treated with thepharmaceutical.

The identification or characterization of pharmaceuticals and modifyingsubstances which is effected using the organisms and methods accordingto the invention can also be effected using substance mixtures, e.g.natural product extracts or fermentation broths, with the substancemixture initially being tested and, when activity has been demonstrated,the mixture then being separated or fractionated into individualsubstances, where appropriate stepwise, in the direction of continuallyincreasing purity, in order to finally arrive at the pure activecompound or the pure modifying substance. Accordingly, during the courseof the separation or fractionation, the method according to theinvention will as a rule be repeated as often as required foridentifying the fraction which in each case contains the active compoundor the modifying substance.

The definitions of the methods according to the invention which arepresented should consequently also encompass substances which arepresent in the form of a mixture with other compounds.

The invention also extends to substances which are effective in thetreatment and/or prevention of neurodegenerative diseases, includingthose in which plaque-like deposits appear in the nervous system, andsubstances which are suitable for modifying the effect of apharmaceutical in the treatment and/or prevention of such diseases,which substances have been identified using one of the methods accordingto the invention which have just been explained. It is possible to usethese substances to produce a variety of pharmaceuticals.

The invention also extends to the use of a substance according to theinvention as a lead substance for developing substances which arederived from it and which are effective in the treatment and/orprevention of neurodegenerative diseases, including those in whichplaque-like deposits appear in the nervous system, or substances whichare suitable for modifying the effect of a pharmaceutical in thetreatment and/or prevention of such diseases.

Proceeding from an appropriate lead substance, the derived substancescan be obtained by means of one or more of the methods of chemicalderivatization which the skilled person customarily employs in thisconnection. Only by way of example, a salt formation, an esterification,an etherification or an amide formation may be mentioned in the case ofa lead substance being present in the form of an organic acid, or theformation of a salt with an organic or inorganic acid may be mentionedin the case of a lead substance which is present in the form of anamine. Hydroxyl groups which are present on the lead substance can beused, for example, for etherification or customary reactions of adifferent nature. The invention also encompasses any types of formationof addition compounds and solvates and of substitutions which can beperformed on the backbone chain of a lead substance, for example on anaromatic ring structure which is present in this chain. Substitutions ofhydrogen atoms, halogen atoms, hydroxyl groups, amine groups, carboxylicacid groups or alkyl groups, or substitutions by such groups or atoms,can, for example, be performed.

Under another aspect, the invention furthermore relates to the use ofthe organisms, according to the invention,

-   -   for identifying and/or characterizing other genes which are        connected with the mechanisms involved in the genesis, the        course and/or the propagation of neurodegenerative diseases,        including those in which plaque-like deposits appear in the        nervous system, in particular Parkinson's disease;    -   for identifying and/or characterizing genes which, in the        mutated or unmutated state, are active in the prevention or        treatment of neurodegenerative diseases, including those in        which plaque-like deposits appear in the nervous system, in        particular Parkinson's disease, and    -   for identifying and/or characterizing genes which, in the        mutated or unmutated state, are able to modify the effect of        pharmaceuticals and/or genes which are employed for the        prevention or treatment of neurodegenerative diseases, including        those in which plaque-like deposits appear in the nervous        system, in particular Parkinson's disease.

In analogy with the search for pharmaceuticals which suppress or augmenta particular phenotype which is associated with neurodegenerativediseases, such as Parkinson's disease, the nematodes according to theinvention, e.g. C. elegans, can also be used for identifying other geneswhich have the same function. A transgenic strain according to theinvention, or a strain which is mutant in one of the candidate genesexemplified above, is used for this purpose. In a following step, thephenotype which is induced by the transgene or the mutation ismanipulated by means of (a) additional mutation(s) in (an) othergene(s). This is effected, as a rule, by means of undirectedmutagenesis, for example by means of using mutagenizing chemicals (e.g.MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; NNG,N-methyl-N′-nitro-N-nitrosoguanidine) or irradiation.

In this way, it is possible to find additional mutations which offset oramplify the phenotype (suppressor or enhancer mutants). The genes whichare affected are candidates for other genes in the same signal pathwayor candidates for genes in parallel signal pathways. Each of these geneswhich have been newly found in this way therefore constitutes anothertarget gene for a pharmacological intervention, for example foridentifying or developing pharmaceuticals (for example possessingactivity toward a product of the gene in question) or for developinggenetic therapeutic agents.

Alongside the pharmacological aspect, this aspect constitutes one of themost important aspects of the animal models according to the inventionand, in particular, of the C. elegans animal model (headword: “pathwaydissection”, identification of the genetic components of a signalpathway).

Accordingly, the invention also encompasses a method for identifyingand/or characterizing genes which, in the mutated or unmutated state,are active in the prevention or treatment of neurodegenerative diseases,including those in which plaque-like deposits appear in the nervoussystem, in particular Parkinson's disease, which method comprises thefollowing steps,

-   -   performing an undirected mutagenesis on organisms according to        the invention,    -   determining any changes in the phenotype, which is associated        with the aberrant or nonexistent expression of the at least one        gene connected with such diseases, as have been exemplified        above, which it may be possible to observe in the organisms        resulting from the mutagenesis, as compared with the starting        organisms which were used for the undirected mutagenesis, and    -   identifying the gene(s) which, as a consequence of its (their)        mutation, is/are responsible for the changes in the phenotype        which were observed in the preceding step.

The invention also relates to a method for identifying and/orcharacterizing genes which are able, in the mutated or unmutated state,to modify the effect of pharmaceuticals and/or genes which are used forthe prevention or treatment of neurodegenerative diseases, includingthose in which plaque-like deposits appear in the nervous system, inparticular Parkinson's disease, which method comprises the steps of:

-   -   performing an undirected mutagenesis on organisms according to        the invention,    -   generating descendants of identical type from the organisms        resulting from the mutagenesis,    -   determining, in the descendants which are in each case        identical, a phenotype which may possibly be present and which        is associated with the aberrant or nonexistent expression of the        at least one gene connected to such diseases, as have been        exemplified above,    -   comparing the phenotype which was determined in the descendants        in the preceding step with the corresponding phenotype of the        starting organisms according to the invention,    -   determining, in the descendants which are in each case identical        and which exhibit the same phenotype as the starting organisms,        changes in the phenotype, which are associated with the aberrant        or nonexistent expression of the at least one gene connected to        such diseases, as have been exemplified above, in the presence        of the pharmaceutical to be investigated or when the gene to be        investigated is expressed, and    -   comparing the changes which have been determined in the        phenotype with the changes in the phenotype of the starting        organisms in the presence of the pharmaceutical to be        investigated or when the gene to be investigated is expressed,        in order to ascertain any differences in the phenotype change        which may possibly be occurring, and    -   identifying the gene(s) which, as a consequence of its (their)        mutation, is/are responsible for the differences in the        phenotype changes which may possibly have been observed in the        preceding step.

The genes in question can be identified in a variety of ways known toskilled persons in this field, for example by means of recombinationanalysis. If the sought-after gene can be restricted to a limited numberof candidate genes, it is also possible, for example, to carry out adirected mutation of corresponding starting organisms and to determinethe phenotype changes or differences in phenotype change resultingtherefrom in order to ascertain whether, after mutation, a candidategene does in fact come into consideration for the phenotype changes ordifferences in phenotype change.

In this connection, it is also possible, for example, for one of thefollowing methods to be used:

-   -   (a) a method for investigating the activity of a gene in the        treatment and/or prevention of neurodegenerative diseases,        including those in which plaque-like deposits appear in the        nervous system, which method comprises expressing the mutated or        unmutated gene in an organism according to the invention and        determining changes in the phenotype which is associated with        the aberrant or nonexistent expression of the at least one gene        connected with such diseases, as has been exemplified above, or    -   (b) a method for investigating the suitability of a gene for        modifying the effect of a pharmaceutical in the treatment and/or        prevention of neurodegenerative diseases, including those in        which plaque-like deposits appear in the nervous system, with        the method comprising,        -   exposing an organism according to the invention to the            pharmaceutical and determining the changes which occur, due            to the effect of the pharmaceutical, in the phenotype which            is associated with the aberrant or nonexistent expression of            the at least one gene connected to such diseases, as have            been exemplified above,        -   exposing at least one further specimen of the organism of            the type used in the preceding step, in which organism the            gene to be investigated is additionally expressed, to the            pharmaceutical, and comparing any changes which may possibly            occur in the phenotype, which is associated with the            aberrant or nonexistent expression of the at least one gene            connected to such diseases, as have been exemplified above,            with the changes in the phenotype which occur due to the            effect of the pharmaceutical on its own.

For the purpose of identifying pharmaceuticals and active substances, itis possible to use the newly identified genes to generate correspondingorganisms which exhibit aberrant or nonexistent expression with regardto those newly identified genes and to investigate pharmaceuticalcandidates entirely in accordance with the above exemplifications.Everything which has previously been stated, for example with regard toorganisms according to the invention, with regard to identifying orinvestigating pharmaceuticals, with regard to producing pharmaceuticals,etc., applies here in a corresponding manner.

Finally the invention also encompasses the use of the specific, C.elegans-derived Parkin gene for producing a gene therapeutic agent forpreventing'or treating neurodegenerative diseases, including those inwhich plaque-like deposits appear in the nervous system (amyloidoses),and, in particular, Parkinson's disease. Such gene therapeutic agentsare used, in particular, when defects or mutations have been identifiedin a particular one of said genes in a patient or an individual who isnot yet exhibiting any symptoms of such a disease and these defects ormutations are known to be connected with the onset, the course or theseverity of such diseases and possibly also the transmission of suchdiseases to descendants.

In addition to this, other aspects of the invention relate to isolatednucleic acid molecules, in particular DNA molecules, but also RNAmolecules, for example, which encode a C. elegans Parkin having theamino acid sequence given in FIG. 3, in particular those molecules whichexhibit a nucleic acid sequence given in FIG. 3. The invention alsoencompasses isolated nucleic acid molecules whose nucleotide sequenceexhibits at least 75%, in particular at least 80%, especially at least85%, preferably at least 90%, particularly preferably at least 95%, andmost preferably at least 98%, sequence similarity to the abovementionednucleic acid molecules, in particular those which hybridize with theabovementioned nucleic acid molecules under stringent conditions.Preferably, such a hybridization takes place under low stringencyconditions while, in another embodiment, it also takes place under highstringency conditions. Within the context of this description, lowstringency conditions are understood as meaning a hybridization in 3×SSCat from room temperature to 65° C. and high stringency conditions areunderstood as meaning a hybridization in 0.1×SSC at 68° C. SSC is theabbreviation for a 0.15 M sodium chloride, 0.015 M trisodium citratebuffer.

The nucleic acid molecules according to the invention which exhibitnucleic acid sequence similarity with the nucleic acid sequence given inFIG. 3 include, in particular, all the allelic variants of the givennucleic acid sequence.

The invention also extends to the nucleic acid molecules which in eachcase have a nucleic acid sequence which is complementary to theabove-exemplified nucleic acid sequences.

In the present connection, the term “isolated nucleic acid molecule”relates to nucleic acid molecules which are present in a form in whichthey are essentially purified from the main quantity of the nucleic acidmolecules of a different nature derived from the parent cells and/orproducer cells. However, preparations of isolated nucleic acid moleculesaccording to the invention can perfectly well contain otherconstituents, such as salts, medium substances or residual constituentsof the producer cells, such as various proteins.

The invention also extends to fragments of the above-exemplified nucleicacid molecules, in particular those which contain nucleic acid segmentswhich encode amino acid sequence regions which are necessary for thefunction of the expression product, i.e. Parkin. In the latter case, theexpression product of such a nucleic acid fragment can exhibit anefficacy/activity which is equivalent to the complete Parkin protein orelse an efficacy/activity which is increased or decreased; in everycase, preference is given to the expression product still being able, tosome degree, to exert the effect(s) exerted by the complete Parkinprotein. In addition, the invention also extends to those fragmentswhich can be used, for example, as highly specific hybridization probes,PCR primers or sequencing primers, or else as “antisense” or “sense”nucleotides for the specific “homology-dependent gene silencing” of theParkin gene. Depending on their purpose, such fragments will encompassat least 15 nucleotides, but frequently 25 or more nucleotides. For suchpurposes, a nucleic acid fragment according to the invention can also,if necessary, possess one or more labeling or detection molecule(s), forexample a digoxigenin molecule or a biotin molecule.

The invention furthermore encompassses constructs, vectors, plasmids,cosmids, bacmids, YACs, BACs, viral genomes or phage genomes whichcontain one of the exemplified isolated nucleic acid molecules. In thepresent case, constructs are understood as being, inter alia:

-   -   constructs which contain the nucleic acid molecules or nucleic        acid molecule fragments according to the invention under control        of a promoter,    -   gene constructs which contain the nucleic acid molecules or        nucleic acid molecule fragments according to the invention fused        to genes or gene segments of another type,    -   constructs which, for example for a use as PCR primers, comprise        nucleic acid fragments according to the invention which have        been supplemented with additional nucleotide sequence segments        which provide suitable restriction sites for a cloning (of the        products resulting from the PCR amplification).

Another aspect relates to the polypeptides or proteins which are encodedby the exemplified nucleic acid molecules, in particular to a C. elegansParkin having the amino acid sequence given in FIG. 3 and topolypeptides or proteins which are derived therefrom by thesubstitution, modification, deletion, insertion or addition of aminoacids and which exhibit at least 75%, in particular at least 80%,especially at least 85%, preferably at least 90%, particularlypreferably at least 95%, and most preferably at least 98%, sequencesimilarity with the abovementioned amino acid sequences. The inventionalso encompasses fragments of the polypeptides or proteins which areexplicitly specified above and, in particular, those which contain aminoacid sequence segments which are essential for the function of the C.elegans Parkin protein. In a preferred embodiment of the invention,these fragments are still able, to some degree, to exert the effect(s)exerted by the complete Parkin protein. The invention also encompassesfusion proteins which contain the polypeptides, proteins or proteinfragments according to the invention fused to a protein of a differenttype or to a protein segment of a different type, for example a markerprotein or indicator protein.

The invention also extends to transgenic organisms, in particularmicroorganisms, e.g. bacteria, viruses, protozoa, fungi and yeasts,algae, plants or animals, and also parts, e.g. cells, and propagationmaterial, e.g. seeds, of such transgenic organisms, which comprise arecombinant nucleic acid sequence, where appropriate integrated into achromosome or else extrachromosomally, which sequence contains a nucleicacid molecule according to the invention, as has just been exemplified,as a transgene.

Under a preferred aspect, the transgenic organisms will also express thepolypeptide or protein, i.e. C. elegans Parkin or a polypeptide orprotein derived therefrom, which is encoded by the abovementionedtransgene.

Particularly interesting uses of the C. elegans Parkin gene, or ofmutants thereof, have already been exemplified in earlier sections ofthis description.

BRIEF EXPLANATION OF THE FIGURES

FIG. 1: Diagram of the structure of the C. elegans Parkin gene togetherwith exons and introns and the location of the deletions in the two KOmutants KO1 and KO2 (location: Parkin gene: 308-2027; KO1 deletion:804-2550 and KO2 deletion: 382-1513).

FIG. 2: Plasmid pLG0126, which contains the C. elegans Parkin promoterand the C. elegans Parkin-GFP fusion construct (SEQ ID NO:3) (promoter:2-654; Parkin gene: 655-2371; exon 1: 655-723; exon 2: 769-914; exon 3:1005-1437; exon 4: 1484-1574; exon 5: 1854-2058; exon 6: 2112-2186; exon7: 2233-2371; GFP: 2414-3133; 3′-UTR of unc-54: 3179-3923; andampicillin resistance gene: 5249-4389).

FIG. 3: Amino acid sequence and cDNA nucleic acid sequence of the C.elegans Parkin coding region (SEQ ID NO:1 and 2); differences in thenucleic acid sequence as compared with the sequence available on theInternet (CAB04599.1, determination nonexperimental) are identified bycapital letters; determination by RNA isolation, cDNA preparation andsubsequent sequencing.

FIG. 4 : (a) Pattern of expression of GFP under the control of the C.elegans Parkin promoter and (b) pattern of expression of the C. elegansParkin-GFP fusion construct (see pLG0126 in FIG. 2) under the control of653 bp of the C. elegans Parkin promoter in C. elegans (cf. experimentalsection 2.). Parkin-GFP is expressed in most neurons. FIG. 4 b showsneurons in the head region (A) and tail region (D) together with theappurtenant Nomarski micrographs (B and E). Parkin-GFP is also expressedin most muscles. (C) in FIG. 4 b shows muscle fibers in the middle ofthe worm.

FIG. 5: Plasmid pLG0082 (SEQ ID NO:4), which contains exon 3 of the C.elegans Parkin gene and was used for the RNAi experiments (cf.experimental section 12.).

FIG. 6: a) Pattern of loading onto the plate in the chemotaxis assay; b)results of the chemotaxis assay in the case of the C. elegans Parkin KO1worms.

FIG. 7: Duration of the development of the C. elegans wilde-type wormsand the Parkin KO1 worms.

FIG. 8: Micrograph of eggs which were laid by Parkin KO2 worms.

FIG. 9: Size of the descendants and number of the eggs which were laidby Parkin KO1 worms.

FIG. 10: Western blot of extracts from wild-type worms (N2) and wormswhich are expressing human α-synuclein A30P, which was integrated intothe genome and under the control of the sel-12 promoter. The differentfractions from the protein extraction are indicated below the blot: HOM,homogenate; RIPA, RIPA-soluble fraction; S1′, first washing step with 5%SDS; S1′″, third washing step with 5% SDS; UREA, urea-soluble fraction;FA, formic acid-soluble frction. Black arrows denote theα-synuclein-specific bands.

FIG. 11: Analysis of transgenic worms which are expressing α-synucleinA30P as an extrachromosomal array under the control of the unc-119promoter. The immunostaining (A) was carried out using the monoclonalantibody 15G7. (B) Expression of GFP under the control of the datpromoter. (C) DAPI staining; (D) Nomarski micrograph.

FIG. 12: Phenotype of transgenic worms which are expressing humanα-synuclein A30P which was integrated into the genome and under thecontrol of the sel-12 promoter. (A) Nomarski micrograph of a worm whosevulva is protruding from the body (B; C) transgenic worm with protrudingvulva and disrupted egglaying, containing juveniles which have alreadyhatched.

EXPERIMENTAL SECTION

1. Bioinformation

The C. elegans homolog of the human Parkin gene was found by using theBLAST program to search the C. elegans sequences which are madeavailable by the National Center for Biotechnological Information NestedPCR primer pairs for identifying deletions were designed using thePrimer3 program from MIT in Cambridge and a window of 3.2-3.3 kb, andsynthesized.

2. Pattern of Expression of Parkin in C. elegans

The expression of the Parkin gene in C. elegans was analyzed using GFPreporter genes (GFP=greeen fluorescent protein).

I. In a first step, the plasmid pBY1013, which contained 4085 bp of theParkin promoter and the GFP gene, was constructed.

4085 bp from the 5′ region of the Parkin locus, amplified as a PCRproduct using the primers

(SEQ ID NO:5) RB850 GGGCCGCGGCATGCGAATACAATGACGTAAGCGACGTGG, (SEQ IDNO:6) RB851 CCCGTCGACTCATCAGACATGCTTCATGAGAGCwere cloned, as an SphI/SalI PCR fragment, into the vector pPD95.75 (Dr.Andre Fire, Carnegie Institution of Washington, Baltimore, Md., USA).

A PCR product obtained from an amplification using the primers

RB853 (SEQ ID NO:7) GGGCCGCGGTTCGAATTTGAAGCTCGCTGCGT RB916 (SEQ ID NO:8)CGCCCGGGAGCTCGTCGACCTATTAAACCAATGGTCCCATTGACACTCwas cloned, as an NheI/SalI fragment, into the GFP vector pBY1023. C.elegans wild-type animals were transformed with the resulting plasmidpBY1013. Semistable transgenic lines were isolated. These exhibited GFPexpression in a large number of neurons and muscles (see FIG. 4 a).

Particular emphasis is to be given to the expression in

-   -   the anal-sphincter and anal-depressor muscles for controlling        defacation,    -   the vulva and uterus muscles for controlling egglaying    -   in the entire pharynx (muscles and neurons),    -   tail neurons, in particular those with a long process in the        ventral nerve cord,    -   head neurons in the region outside of the pharynx.

II. Preparing the plasmid pLG0126 (contains 653 bp Parkinpromoter+Parkin gene+GFP gene as a fusion construct)

3432 bp from the 5′ promoter region of the Parkin gene were excised frompBY1013 by means of inverse PCR and filled in once again using Klenowpolymerase. The 1717 bp Parkin gene was then cloned into the resultingplasmid downstream of the Parkin promoter and upstream of the GFP (inthe same reading frame as the Parkin gene). The resulting plasmidpLG0126 (FIG. 2) was transformed into C. elegans wild-type animals.Semistable transgenic lines were isolated. As in the case of plasmidpBY1013, these lines exhibited GFP expression in a large number ofneurons (e.g. head, tail and pharynx) and muscles (e.g. pharynx, vulvaand uterus) (see FIG. 4 b).

3. Preparing C. elegans Eggs

Approx. 8,000 young adult wild-type animals were rinsed with M9 buffer(22 mM KH₂PO₄, 42 mM Na₂HPO₄, 86 mM NaCl, 1 mM MgSO₄) from the agarplates and collected in 15 ml Falcon tubes until all the worms had beendetached. After that, the worms were centrifuged for 5 min-and thesupernatant was aspirated off. The worm pellet was taken up in 10 ml ofM9 buffer and centrifuged once again at 3000 rpm for 3 min. After thelatter procedure had been repeated, 10 ml of bleach solution (1.44%NaClO, 0.25 M KOH) were added to the worm pellet. After shaking for 4min, the egg preparation was centrifuged at 1,500 rpm for 30 s and thesupernatant was aspirated off and the egg pellet taken up in 10 ml of M9buffer. After that, the pellet was washed 5 times with in each case 10ml of M9 buffer and in each case centrifuged at 3,000 rpm for 1-2 min.

4. Preparing Mutagenized Nematode Worms

In order to prepare the mutagenized nematode worm strains, the eggpellet which resulted from the egg preparation was taken up in 10 ml ofM9 buffer and shaken in an overhead shaker at 20° C. for 20 h. On thefollowing day, the hatched L1 larvae (in each case 6,000 per plate) weresown on 8 plates (OP50 bacteria-inoculated agar plates of 9 cm diameter)and incubated at 15° C. for 60-70 h. After the animals had been rinsedoff with M9 buffer and collected in Falcon tubes, they were washed in M9buffer until the supernatant was no longer opacified by bacteria(centrifugations were in each case at 1,000 rpm for 3 min).Subsequently, 10,000-15,000 animals were transferred into a new Falcontube and treated with trimethylpsoralen (30 μg/ml). The mixture wasincubated for 15 min on the overhead shaker in the dark and, after theworms had settled after 3 min, they were added dropwise to a dry agarplate. After the worms had been allowed to dry for 2-5 min, they wereirradiated for 1 min at 365 nm with a dose of 540 μW/cm². After that,the worms were rinsed off with M9 buffer and centrifuged down and thepellet was added to 3 new plates (9 cm in diameter and inoculated withOP50 bacteria). After the plates had been incubated at 20° C. for 24 hin the dark, an egg preparation was carried out (see above) andincubated overnight at 20° C. After that the synchronized andmutagenized F1/L1 animals were centrifuged down and taken up in Scomplete medium (200 mM NaCl, 50 mM KH₂PO₄, 13 mM cholesterol, 10 mMsodium citrate, 3 mM CaCl₂, 3 mM MgSO₄, 100 U of nystatin/l, 100-foldPSN antibiotic mix, trace elements, HB101 bacteria having an OD_(600 nm)of 2.0) (400 worms/ml). The worm suspension was sown in 17-33 microtiterplates at the rate of 50 μl per well and the plates were incubated at20° C. for 5-6 days. After in each case one quarter (12.5 μl per well)of the worm suspension had been removed for preparing the genomic DNAand the well lysates, the worm plates were placed in storage boxes forlong-term storage, sealed with parafilm and incubated at 15° C.

5. Preparing Pooled Genomic DNA

In each case one quarter of the worm suspension (12.5 μl per well) fromall the 96 wells from an original plate were combined in a 15 ml Falcontube and 1 ml of M9 buffer was added. The Falcon tubes were washed twicewith in each case 10 ml of M9 buffer (centrifugation at 3,000 rpm for 3min), after which 4 ml of washing buffer (20 mM tris buffer, pH 7.5,containing 100 mM NaCl and 50 mM EDTA) were added. After acentrifugation at 3,000 rpm for 3 min, all but 1 ml of the supernatantwas aspirated off and the remaining supernatant, together with thepellet, was transferred to an Eppendorf tube using a Pasteur pipette.The mixture was centrifuged at 14,000 rpm for 1 min in an Eppendorfcentrifuge and all but 250 μl of supernatant was taken off. 350 μl ofwashing buffer were added to this remaining 250 μl and the whole wasfrozen at −80° C. After rethawing, 10 μl of 10% SDS, 2 μl of proteinaseK (20 mg/ml) and 1 μl of β-mercaptoethanol were added, after which thewhole was mixed by tipping and then digested at 65° C. for 1 h. Onceagain, 2.5 μl of proteinase K (20 mg/ml) were added and the mixture wasincubated for 1 h. After that, 2.4 μl of RNAse A (10 mg/ml) were added,and the whole was mixed by tipping and then incubated at 37° C. for 15min. After 250 μl of protein precipitation solution (Promega) had beenadded, each Eppendorf tube was vortexed for 10 s, placed on ice for 5min and centrifuged (at 14,000 rpm for 10-15 min, if possible whilebeing cooled). The supernatant, which contained the DNA, was carefullytaken off and added to a new Eppendorf tube. 500 μl of isopropanol wereadded to these supernatants, after which mixing was carried out bytipping and the tubes were then placed on ice for 5 min and subsequentlycentrifuged at 14,000 rpm for 10-15 min. The supernatant was taken offand discarded. 500 μl of 70% ethanol were added to the pellet, whichcontained the DNA, after which the tubes were mixed by tipping andcentrifuged at 14,000 rpm for 10 min. The supernatant was very carefullypipetted off. The remainder of the ethanol was evaporated off by placingthe open Eppendorf tubes in a heating block at 37° C. The pellet whichremained was taken up in 350 μl of 10 mM tris, 1 mM EDTA and left tostand at room temperature overnight. On the following day, the genomicDNA was placed in a heating block at 50° C. for 1 h and distributed onPCR plates.

6. Preparing Well Lysates

Well lysates were prepared from a quarter of the sown-out wormsuspension (12.5 μl ) by 12.5 μl of the worm suspension being taken upin 12.5 μl of lysis buffer (20 mM tris buffer, pH 8.2, containing 100 mMKCl, 5 mM MgCl₂, 0.9% NP-40, 0.9% Tween-20, 0.02% gelatin, 14.4 μg/ml ofproteinase K), being digested at 65° C. for 6 h, then being incubated at95° C. for 15 min in order to inactivate the protein kinase K, and thenbeing frozen down at −80° C. after having been cooled briefly.

7. Using Nested PCR to Identify Deletions

Nested PCR was used to test genomic DNA plate pools and well lysates fordeletions. In nested PCR, a PCR is first of all carried out using twoexternal primers and a PCR is then carried out on the resulting productusing two internal primers, in order to obtain higher specificity andbetter amplification. The genomic DNA was stamped directly, using asample stamper (V & P Scientific, San Diego, USA), into a mixture(reaction volume of 25 μl) which contained Taq polymerase (Hoffmann-LaRoche, 0.5 units/reaction), dNTP mixture (0.2 mM) and primers (0.4 μM).The reaction ran for 35 cycles at 92° C. for 20 sec, 56° C. for 1 minand 72° C. for variable times (15-120 s) in Perkin-Elmer machines of the9700 type.

8. Analyzing the Deletions

PCR products were analyzed using agarose gels (1% agarose), andsequenced when identifying desired deletions.

9. Isolating the Mutants

The worms present in the well which was identified as containing wormshaving the deletion in the target gene, i.e. having shorter PCR productsthan in the case of the wild-type worms, were taken out of the liquidculture and added individually to agar plates. After havingself-replicated successfully, the parent animals were analyzed forhomozygous and heterozygous worms using various primers which lay withinand outside the deletion. Positive strains, i.e. worm strains havingdeletions in the target gene in either one (heterozygous) or both(homozygous) alleles were outbred five times with wild-type worms inorder to eliminate other potential mutations. Homozygous strains whichwere outbred four times were used for the subsequent experiments. Themutants depicted in FIG. 1, i.e. Parkin KO1 (strain designation XY1003)and Parkin KO2 (strain designation XY1046), were obtained in thisconnection.

10. Phenotypinq the Parkin knock-out mutants KO1 and KO2

a) Chemotaxiassay

Method:

Four plates composed of 1.6% agar, 5 mM KH₂PO₄, 1 mM CaCl₂ and 1 mMMgSO₄ were used per worm strain to be tested. The plates were divided upas shown in FIG. 6 a).

1 μl of NaN₃ (1 M) was in each case applied at (+) and (−).Subsequently, 1 μl of the previously diluted attractant was applied at(+) while 1 μl of EtOH (100%) was applied at (−). The worms werecultured with E. coli on eight large agar plates, then washed off withH₂O and Tween, washed 1×at 1000 rpm and taken up in 1 ml of H₂O andTween. 10 μl of the worm suspension (approx. 100 worms) were applied toeach test plate. By holding the test plate vertically, the worms weredistributed over a line which resulted in identical distances to theattractant and a negative control being achieved.

The attractants used were isoamyl alcohol (1:200), diacetyl (1:1000),pyrazine (10 mg/ml) and 2,4,5-trimethylthiazole (1:1000). All thesubstances were diluted in 100% EtOH. The assay was carried out at roomtemperature for 90 min. After that, chloroform was added to the lids ofthe test plates in order to immobilize the worms.

Evaluation:

The worms which were present within a radius of 1.5 cm around theattractant (A) or round the negative control (B) after the time hadexpired were counted. The chemotaxis index was calculated from thesevalues as follows:

$\frac{A - B}{A + B}$Results:

The result is shown in FIG. 6 b).

The Parkin KO1 worm strain did not react to the attractant diacetyl(odr).

The Parkin KO2 worm strain behaved in a similar manner (results notshown).

b) Period of Development

Method:

10 eggs of the worm strain to be tested were in each case tipped onto asmall plate. The time which elapsed until the first eggs were once againlying on the plate was determined.

Results:

In the case of the wild-type, 100% of the worms had laid their firsteggs once again after 72 hours. In the case of the Parkin KO1 wormstrain, the first eggs had only been laid after more than 100 hours. TheParkin KO1 development period was therefore retarded, as shown in FIG.7.

c) Analysis of Freshly Laid Eggs

Method:

Three adult worms were transferred to a fresh plate. After from 30 to 60min, it was possible to use Nomarksi microscopy to observe the recentlylaid eggs. In the case of the wild-type worm strain, the eggs were laidduring gastrulation (28 cells) or up to a cell stage consisting of 50cells (beginning of morphogenesis). FIG. 8 shows two eggs which werelaid by Parkin KO2 worms.

Results:

Some of the eggs laid by the Parkin KO2 worm strain are in a cell stagewhich is too far advanced. Parkin KO2 consequently suffers from apartial egglaying defect (egl). This defect was also observed in thecase of the Parkin KO1 worm strain.

d) Number of Descendants

Method:

10 eggs were isolated individually per worm strain tested. As soon asthe individuals developing from these eggs laid eggs once again, theworms were changed in a 24-hour rhythm until no more eggs were laid. Thedescendants were counted in the L1/L2 stage. In this way, it waspossible to count all the descendants. Furthermore, it was possible tooutline the chronological course of the egglaying in the sameexperiment.

Results:

The result is shown in FIG. 9.

e) Determining the Sinusoidal Curve for Testing Mobility

Method:

5 young adult worms were deposited on a fresh plate. The amplitude andsinusoidal length of the sinusoidal track left behind in the bacteriallawn were then measured by way of a visual display. 10 tracks weretraced on film, which was then overlaid.

Results:

The sinusoidal amplitude in the case of the Parkin KO1 and KO2 wormstrains corresponded approximately to the wild-type amplitude. However,the sinusoidal length was approx. 20% shorter than in the case of thethe wild-type worm strains. The mobility of the Parkin KO1 and KO2 wormstrains was consequently disrupted (unc).

f) Defacation

Method:

The periods between the expulsion and between pBoc (intestinalcontraction shortly before the expulsion) and the subsequent expulsionwere measured. 10 animals were observed per worm strain.

Results:

Both the expulsion-expulsion cycle and the pBoc expulsion cycle wereshorter in Parkin KO1 and Parkin KO2 than in the wild type.

11. Complementing the C. elegans Gene with the Homologous Human Gene

In order to carry out the detailed molecular characterization of themutants, they were subjected to a function test. Tests were carried outto determine whether the phenotypes really were to be attributed to thedeletion in the target gene, on the one hand, and, on the other hand,whether the C. elegans target gene and the human target gene werefunctionally conserved. For this reason, the deleted C. elegans targetgene itself, on the one hand, and the homologous human gene, on theother hand, were in each case inserted into the C. elegans knock-outstrains possessing the deletions in the target genes. Both genes wereemployed as cDNA constructs containing the C. elegans promoter (FIG. 2)in order to ensure a pattern of expression which corresponded to that ofthe target gene. An is injection microscope (Zeiss) was used tointroduce the constructs directly into the worm as plasmids (50 ng/μl).The defects in chemotaxis, development, egglaying, defacation andmobility were able to be reversed both by introducing the C. elegansParkin and by introducing the human Parkin.

12. RNAi Experiment Using the C. elegans Parkin Gene

For the purpose of carrying out an RNAi feeding experiment forinactivating the Parkin gene, the plasmid pLG0082 (see FIG. 5) was firstof all constructed: a 441 bp DNA fragment constituting a part of exon 3of the Parkin gene was prepared by PCR amplification using theoligonucleotides 1-RNA1 and 1-RNA2. The choice of the oligonucleotidesresulted in the introduction of an NcoI restriction cleavage site at the5′ end and a Pstl restriction cleavage site at the 3′ end. After the PCRfragment had been isolated, it was cut with the correspondingrestriction enzymes and ligated into the vector pPD129.36 (Dr. AndrewFire, Carnegie Institution of Washington, Baltimore, Md., USA), whichhad been digested with the same enzymes. In this way, the Parkinfragment was positioned between two T7 RNA polymerase promoters.

The E. coli strain HT115 (Dr. Lisa Timmons, Carnegie Institution ofWashington, Baltimore, Md., USA) was transformed with the plasmidpLG0082 and ampicillin-resistant colonies were isolated. This straincarries a chromosomal copy of the gene for T7 RNA polymerase under thecontrol of a lacZ promoter and therefore produces both sense RNA andantisense RNA corresponding to exon 3 of the Parkin gene followinginduction with 1 mM IPTG. Growth of the worms on these bacteria leads tothe worms taking up the double-stranded Parkin RNA and to the Parkingene being inactivated as a result.

Per experiment, five wild-type worms in the L3 stage of development werereared on these feed bacteria at 15° C. for about 2-3 days. Following achange to 20° C., their descendants were subsequently analyzedphenotypically.

1-RNA1: CAGACAAACCATGGTTCTCC (SEQ ID NO:9) 1-RNA2: CTTACTCTGCAGCAGAATTGG(SEQ ID NO:1O)

Defects in egglaying, defacation and mobility were observed.

13. Preparing a C. elegans Mutant which Expresses Human α-synuclein

Cloning the Constructs

In order to prepare the constructs which express α-synuclein under thecontrol of the unc-119 promoter, human α-synuclein cDNA (Dr. ChristianHaass, Ludwig-Maximilians University Munich) and the cDNAs of the A53Tand A30P mutants (Dr. Christian Haass, Ludwig-Maximilians UniversityMunich) were amplified by PCR using primers which contained cleavagesites for SalI. The amplified and cut DNA fragments were cloned into theSalI cleavage sites of the vector pPD49.24 (Dr. Andrew Fire, CarnegieInstitution of Washington, Baltimore, Md., USA), into which the promoterof the C. elegans unc-119 gene had previously been introduced. The 5′promoter region upstream of the unc-119 gene had been amplified by meansof PCR and two primers from the cosmid M142. This gave rise toconstructs pBY456 (unc-119::α-synuclein wt), pBY457(unc-119::α-synuclein A53T) and pBY458 (unc-119::α-synuclein A30P).

In order to prepare the constructs which express α-synuclein under thecontrol of the sel-12 promoter, human α-synuclein cDNA and the cDNAs ofthe A53T and A30P mutants were excised from the constructs pBY456,pBY457 and pBY458, respectively, using the restriction enzymes Mscl andNcoI and cloned into the Mscl/NcoI incision sites of the vector pPD49.26(Dr. Andrew Fire, Carnegie Institution of Washington, Baltimore, Md.,USA), into which the promoter of the C. elegans sel-12 gene havepreviously been introduced. The 5'promoter region upstream of the sel-12gene was amplified by means of PCR and the two primers RB759 and RB110(see below) from the cosmid C08A12. This gave rise to the constructspBY1158 (sel-12::α-synuclein wt), pBY1159 (sel-12::α-synuclein A53T) andpBY1160 (sel-12::α-xynuclein A30P).

RB759: CCCGGCTGCAGCTCAATTATTCTAGTAAGC (SEQ ID NO:11) RB110:GTCTCCATGGATCCGAATTCTGAAACGTTCAAATAAC (SEQ ID NO:12)

The unc-119 promoter gave rise to panneuronal expression whereasexpression in the case of the sel-12 promoter took place neuronally andin some muscle cells.

Preparing Transgenic Animals

Transgenes were introduced into C. elegans by microinjecting them intothe gonads (Mello et al., 1992). The above-described α-synucleinconstructs were coinjected together with the marker plasmids pBY1153(sel-12:: EGFP) and pBY266 (dat::EGFP), respectively, and GFP-expressingdescendants were selected.

Strains which possess the transgene stably integrated in the chromosomewere obtained by X-irradation (5000 rad) of lines which harbored thetransgenic, extrachromosomal arrays. The descendants of the X-irradiatedanimals were screened for 100% transmission of the transgenic marker. Inorder to eliminate any possible background mutations, the integratedlines were backcrossed with wild-type worms (N2).

PAGE and Immunoblotting

The proteins were extracted from C. elegans, and PAGE and Westernblotting were carried out, as described by Okochi et al. (2000). Themonoclonal antibody 15G7 (Connex, Martinsried) was used, diluted 1:4,for immunostaining of the Western blots. The secondary antibody employedwas a peroxidase-conjugated goat a rat antibody (Jackson) which wasdiluted 1:5000.

Immunostainings

The immunostainings were carried out in accordance with a standardprotocol (Finny et al. 1990) using the monoclonal antibody 15G7 (Connex,Martinsried) in the undiluted state. The secondary antibody employed wasa Texas red-coupled goat α rat antibody diluted 1:2000.

α-Synuclein Aggregations in Urea Extracts in Vitro

In a Western blot (FIG. 10) using a monoclonal antibody directed againstα-synuclein, yeast extracts of proteins from the integratedsel-12/α-synuclein A30P worm exhibit a high molecular weight band whichis not visible in N2 worm extracts which have been treated in the sameway.

α-Synuclein Aggregations in Neurons in Vivo

The immunostainings (FIG. 11) of unc-119/α-synuclein A30P using amonoclonal antibody directed against α-synuclein exhibit clear signals,both in the cell body and in axons, with pearl necklace-like stainingfrequently being seen in the latter. This points to the α-synucleinprotein being transported to the presynapse and to the protein beingaccumulated in the axons.

Defects in Egglaying and in Vulva Formation

Worms in which the sel-12/α-synuclein A30P construct is stablyintegrated into the genome exhibit deformation of the vulva (p-vul) anda defect in egglaying (egl) (FIG. 12).

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1. A genetically modified nematode belonging to genus Caenorhabditiscomprising Parkin gene K08E3.7, wherein the expression of said Parkingene is nonexistent.
 2. The genetically modified nematode as in claim 1,wherein the nonexistent Parkin gene expression produces plaque-likedeposits in the nervous system of the genetically modified nematode. 3.The genetically modified nematode as in claim 1, further comprising aphenotype associated with the nonexistent expression of said Parkin geneselected from the group consisting of: a defect in chemotaxis; a defectin egglaying; an extended period of development; a decreased number ofdescendants; problems in coordination; a defect in defecation; retardedlocomotion; and a reduced body length.
 4. The genetically modifiednematode as in claim 1, wherein said Parkin gene is partially orentirely deleted.
 5. The genetically modified nematode as in claim 1,wherein said Parka gene is mutated.
 6. The genetically modified nematodeas in claim 1, wherein said Parkin gene has been inactivated at leasttransiently by means of the RNA interference technique.
 7. Thegenetically modified nematode as in claim 1, further comprising atransgene.
 8. The genetically modified nematode as in claim 1, whereinsaid nematode belongs to the species selected from the group consistingof: elegans, vulgaris, and briggsae.
 9. The genetically modifiednematode as in claim 1, wherein said nematode is Caenorhabditis elegans.